Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARS) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (June et al., 2011). However, the particular combination of signaling domains, transmembrane and co-stimulatory domains used with respect to CD19 ScFv, was rather antigen-specific and cannot be expanded to any antigen markers.
According to the data from the Centers for Disease Control and Prevention. [http://www.cdc.gov/cancer/colorectal/statistics/race.htm], incidence of colorectal cancer in the US population over the year 2011 was about 50 per 100 000 people for women and up to 60 for males in the black people population, leading to 50% mortality. This incidence has only decreased by 10% over the last decade.
One candidate antigen of immunotherapies for solid tumors, including the colorectal, ovarian and gastric and also for non-solid tumors such as childhood acute lymphoblastic leukemia (ALL) is the trophoblast glycoprotein, also known as TPBG or 5T4 (UniProt: 013641). 5T4 is often referred to as an oncofetal antigen due to its expression in foetal trophoblast (where it was first discovered) or trophoblast glycoprotein (TPBG). 5T4 protein is an N-glycosylated transmembrane 72 kDa glycoprotein containing seven leucine-rich repeat regions (Hole et al, 1988). The 5T4 antigen was found to be expressed in number of carcinoma including gastric (Starzynska et al. 1995), ovarian and carcinoma (Wrigley et al. 1995). Also, 5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia (Castro et al. 2012). It has very limited expression in normal tissue but is widespread in malignant tumors throughout their development (Cars berg et al. 1995).
The present inventors have thus considered that 5T4 could be a valuable target antigen for treating solid tumors such as colorectal, ovarian and gastric tumors, by using CAR-expressing T cells.
As an alternative to the previous strategies, the present invention provides with 5T4 specific CARs, which can be expressed in immune cells to target 5T4 malignant cells with significant clinical advantage.
There is still the need for the improvement of CAR functionality by designing CAR architecture and using suitable components since these parameters play a role important and a fine tuning may be necessary.
The inventors have found that, by combining CAR architecture to the choice of suitable components, they could obtain specific 5T4 single chain CARs with high cytotoxicity towards cancerous target cells.